Location: Corn Insects and Crop Genetics Research2010 Annual Report
1a. Objectives (from AD-416)
The fundamental goal of this project is to provide researchers who have interests in the genetics and genomics of western corn rootworm (WCR) with a set of resources that will accelerate ongoing and future research programs. During the past half-decade, substantial progress has been made in the study of this species, particularly in the development of Expressed Sequence Tag (EST) data and polymorphic molecular markers. However, these resources have not yet been integrated to the extent that is possible and desirable. This proposal seeks to bring together and expand existing resources via the following objectives: 1) Validate a large number of putative Single Nucleotide Polymorphisms (SNPs) already identified in existing EST data. 2) Construct a comprehensive linkage map of the western corn rootworm genome using validated SNPs and existing microsatellite markers. 3) Associate SNP loci on the linkage map with bacterial artificial chromosome (BAC) clones. 4) Fingerprint SNP-associated BAC clones by restriction mapping and assemble them into contigs. 5) Sequence representative BAC clones from the assembled contigs to characterize the genomic regions surrounding SNP sites. 6) Disseminate the data to the wider research community via a user-friendly website that can accommodate new data in the future.
1b. Approach (from AD-416)
We will take a mass-screening approach for the validation of EST-derived SNPs. This will be done using the Illumina Golden Gate assay platform. Up to 3,072 candidate SNPs with a polyphred score greater than or equal to 95 for which an assay can be designed will be tested for polymorphism against samples of WCR populations from Illinois and Iowa. Markers that are polymorphic in these populations will then be used to genotype the backcross pedigrees to verify that they are Mendelian single-locus markers. DNA extractions from the backcrosses will be prepared at the start of the project so that they are available as soon as they are required. Genotyping of the backcrosses with microsatellites will be done simultaneously with the SNP genotyping. Once genotyping of the backcross pedigrees is completed, linkage maps will be constructed for each pedigree and then merged to form a combined map. The combined map will then be used to select a set of EST sequences associated with validated, mapped SNPs that will be used to design PCR primers to screen the BAC library. BAC clones that are identified as harboring SNP loci will be mapped by restriction digestion and assembled into contigs. Representative BAC clones from each contig will then be sequenced using high throughput 454 pyrosequencing. The BAC clones to be sequenced will be selected based on their location within the SNP-associated contig to maximize the amount of sequence data obtained on both sides of the SNP marker. The genetic maps and associated molecular and other data that will be generated by this project are generally similar in both content and scope to those in other genetic/genomic databases, and in particular to SoyBase. This will allow us to develop prototype databases and web displays in advance of the actual data generation. During the initial development, we will identify a small group of external testers who will be asked to provide input on functionality, displays, etc. This strategy of early development will allow us to release "WCRbase" to the research community as soon as a useful body of data is available, and then extend it as additional data are generated.
3. Progress Report
The goal of this project is to use next-generation genomic sequencing technology (Illumina GoldenGate assay) to identify and verify large numbers of molecular genetic markers called single nucleotide polymorphisms from western corn rootworm. This type of genetic marker (single nucleotide polymorphisms) will be used to create a linkage map that will show the relative position of genes. The closer genes are, the more likely they will be inherited together. Some of these genetic markers can also be useful to understand the structure of an insect population. Verification of these markers is underway. Of the 2,222 candidate markers that we determined to be potentially useful, 1,536 were chosen for further testing in collaboration with the University of Illinois W. M. Keck Center for Comparative and Functional Genomics. A western corn rootworm bacterial artificial chromosome library was constructed. A bacterial artificial chromosome is a type of tool, called a vector, that inserts long stretches of DNA from any species into a bacterium. Once the foreign DNA has been cloned into the bacterium, many copies of it can be made and sequenced. This library was screened by us at the Corn Insects and Crop Genetics Research Unit, Ames, Iowa, and collaborators at the University of Nebraska to identify genes coding for important proteins such as receptors and enzymes that are often involved in insecticide resistance. DNA from ten of these clones has been extracted, and is now being purified and prepared for sequencing. In addition to providing direct data about the target genes, information from sequence analysis will be useful in characterizing the overall structure of the western corn rootworm genome in preparation for future projects. The library will be screened further with the single nucleotide polymorphism markers once they are validated, and 80 will be strategically selected for full sequencing to facilitate linkage mapping. DNA from laboratory colonies of insects, and from a number of wild populations, including western corn rootworms resistant to control by corn-soybean planting rotation, has been extracted and is ready for linkage mapping and genome scanning experiments. The genome scan will be used to identify genetic markers linked to rotation resistance genes. The size of the western corn rootworm genome was determined using flow cytometry (a technique for counting microscopic particles) and is very large. Progress of University of Illinois and University of Nebraska collaborators is monitored through frequent email correspondence, telephone calls initiated by both parties, conference calls, and discussions at jointly attended technical meetings and conferences.